Hibit control protein

The HiBiT Control Protein (Promega, Madison, WI, USA) is a 20 μM solution of purified recombinant 36 kDa HaloTag^® protein fused at its carboxy terminus to the 11-amino-acid HiBiT tag. The 20 μM HiBiT Control Protein was diluted 2000 times with the supernatant from the starch fermentation medium to obtain a solution of 10⁻⁸ M. Nine gradients were then diluted between 10⁻⁸ M and 10⁻⁹ M. The luminescence of each gradient was then detected to plot the calibration curve. A Tris-tricine-SDS-PAGE gel preparation kit (Solarbio, Beijing, China) was used for protein separation of the shake flask supernatant. After monellin was separated using Tris-tricine-SDS-PAGE, it was transferred to a PVDF membrane at 350 mA for 30 min. Protein blotting was performed using the Nano-Glo^® HiBiT Blotting System (Promega, Madison, WI, USA) according to the manufacturer’s instructions, and then the PVDF membrane was transferred to a gel multifunction imager and imaged after 5 min of exposure.

As a proxy to measure extracellular toxin release, C. albicans strains were engineered to express a HiBiT-tag at the C-terminus of candidalysin. The 11 amino acid HiBiT tag encodes for a small domain of NanoLucⓇ luciferase (Promega). C. albicans cells were grown in yeast nitrogen base (YNB) O/N, washed 3X in PBS, counted, diluted to a final density of 5x10⁵ cells/mL in MOPS-buffered RPMI-1640, and incubated at 37°C with shaking (200 rpm) for 16 h. The following day, cultures were centrifuged at high speed to pellet cells and cell-free supernatants isolated. The Nano-GloⓇ HiBiT Extracellular Detection System was used according to the manufacturer’s instructions (Promega). Relative light units (RLU) were measured using a BioTek Synergy plate reader using an integration time of 2 s. RLUs were converted to concentrations by extrapolating to a standard curve generated using the HiBiT Control Protein (Promega).